Acute and chronic antibody-mediated rejection are major problems for transplant recipients because current immunosuppressive regimens are insufficient to control humoral immunity against the graft. Our preliminary data in the pre-clinica swine model not only demonstrate induction of stable B-cell tolerance following donor hematopoietic cell infusion but the elimination of donor-reactive antibody production in sensitized recipients. This proposal is designed to determine the mechanistic basis for induction of stable B-cell tolerance to transplantation antigens in order to facilitate development of clinically relevant approaches to overcome humoral immunity to transplants. This project is based on findings that donor cells can induce stable B-cell tolerance, independent of T-cell tolerance, when infused intravenously in MHC-mismatched swine recipients. Exposure to donor cells following partial T-cell depletion, very low-dose total body irradiation, and a short course f cyclosporine mono-therapy, results in inability of swine recipients to generate donor specific alloantibody responses, even after multiple immunizations with donor cells. The ability to generate antibody responses to vaccine and protein antigens remains intact in these animals. Pre-existing anti-donor antibody could no longer be detected following donor cell infusion under the mild conditioning described above and alloantibody could not be induced, despite clear lack of T-cell tolerance. These preliminary data demonstrate that it may be possible to specifically eliminate anti-donor antibody production in pre-sensitized patients prior to organ transplantation. We hypothesize that pre-existing alloreactive host B cells are tolerized following exposure to donor cells under immunosuppressive conditions that sufficiently reduce T-cell alloreactivity, yet preserve immune function. We also hypothesize that B-cell tolerance is maintained by an active regulatory mechanism preventing germinal center formation in response to repeated exposures to donor cells without immunosuppression. These hypotheses will be addressed through the following aims: 1) Determine the mechanistic basis for induction and maintenance of B- cell tolerance to donor cells; and 2) Examine conditions which reliably eliminate anti-donor antibody production in highly sensitized recipients. Understanding the mechanisms involved in inducing and maintaining B-cell tolerance in a clinically relevant large animal model will facilitate design of appropriate clinical conditioning protocols that enhance or at least do not inadvertently destroy these mechanisms. Although murine models have been critical to understanding transplantation biology, large animal models are essential in facilitating clinical translation of novel findings in this field. Protocols developed in swine have potential for direct translation to the clinic, and to date, all have been translatable to non-human primates and/or patients. Since treatment of the sensitized patient remains an important, unmet need in the field of transplantation, results of these studies could lead to a breakthrough in improving transplant outcomes, particularly in highly sensitized patients.